In the early 1990s, amid great excitement, six trials took place of transplants of immature muscle cells from healthy relatives into boys with Duchenne muscular dystrophy. (Five were funded by MDA.)

The strategy involved intramuscular injection of a type of muscle repair cell called a satellite cell, multiplied many times (cultured) in laboratory containers. The cells also were called “myoblasts,” meaning they give rise to muscle cells. (“Blast” comes from the Greek word for germ or bud, and “myo” from the Greek word for muscle.)

The technique, myoblast transfer, looked promising in mice. Unfortunately, the results in humans were less than impressive.

Almost all the transplanted cells died within a few days, none migrated very far from the site where they were injected, and almost no dystrophin, the protein missing in boys with DMD, was produced in the recipients.

Later analyses determined that there were at least three hurdles to be cleared before myoblast transfer could be successful: Immune-system rejection of the myoblasts had to be prevented; the limited mobility of the myoblasts had to be increased; and the ability of the myoblasts to fuse with existing muscle fibers had to be improved.

It seemed the end of cell transplantation as a treatment for muscular dystrophy, and many of its former proponents turned their attention to gene insertion therapy.

But not everyone gave up on trying to surmount the obstacles involved in putting new cells into the body as a treatment for muscle disease.

Modifying myoblasts

Which muscle progenitor cells are best for transplantation?

Embryonic stem cells may be too immature (undifferentiated) for transplantation. A major risk is that they can form tumors.

Myoendothelial cells, mesoangioblasts and pericytes, which are in or around blood vessels, and SP cells, which are thought to be between the muscle fibers, look like good potential candidates for transplantation.

Satellite cells (myoblasts), after they’ve been grown in the laboratory, may be too mature to yield the desired effects after transplantation, although modifications to these cells could change that.

One researcher who continued work on myoblast transplantation is Jacques Tremblay, a professor of medicine at Laval University in Quebec City. (MDA has funded some of Tremblay’s work.)

Tremblay believes myoblasts, if properly selected and modified and if conditions are right, still are a viable treatment for DMD and perhaps other forms of muscular dystrophy.

He takes satellite cells from muscle biopsies of close relatives of DMD patients and cultures them in the lab. “When they proliferate, we call them myoblasts,” he says. “We can make 20 million in three weeks from a single donor satellite cell.”

“There are two conditions where myoblasts will fuse into muscle,” Tremblay says. “Either the muscle fiber is damaged, or the muscle fiber is undergoing hypertrophy [enlargement].”

Muscle damage in MD is a constant occurrence, so the first condition is easily met. Tremblay is attempting to induce the second condition — muscle-fiber hypertrophy — by manipulating a growth-limiting protein called myostatin. “By blocking the myostatin signal,” he says, “you’re inducing muscle hypertrophy.” Doing this at the time of myoblast transfer in mice improves the success of the procedure, he reports.

Tremblay’s group is investigating exactly how transplanted cells die and trying to reduce cell death. Inflammation and lack of oxygen (hypoxia) are two major problems, he says. “If you make the pockets of myoblasts a bit too big, myoblasts in the center will die of hypoxia. They have to be distributed very finely along the injection trajectories.”

The group is working on genetically modifying myoblasts to increase their hardiness, but Tremblay says it will be difficult to get this type of strategy accepted as safe by regulatory agencies.

In studies funded by MDA, he found that simply heating the myoblasts for a few hours improved their survival after transplantation. The heat treatment, he notes, “is something we could introduce in the next transplantation that we will do in patients.”

He’s also using a technique he calls high-density injection, which allows millions of cells per square centimeter (0.4 inches) to be injected at a time, with several injections.

This technique was recently tried in a 26-year-old man with advanced DMD, who tolerated the procedure well and experienced some increased mobility in a thumb.

Tremblay’s next step is a trial of this approach in 10 adult men with DMD or Becker MD (BMD). The trial has run into some obstacles with Canadian regulatory agencies, but he hopes to be able to begin soon.

As for dampening the immune response, Tremblay believes there are now better drugs and better strategies than there were when the original myoblast transfer trials were conducted.

He likes a drug called tacrolimus (Prograf), which is used to prevent rejection of transplanted organs, but says giving patients their own cells with a dystrophin gene inserted might be the best way to ensure acceptance by the immune system.

Since the cells would get their new genes while outside the body, the strategy might be safer than gene therapy, in which new genes, usually inside viral shells, are injected directly into the patient, as it avoids exposing the patient directly to the virus.

“Using original satellite cells might be better [than multiplying them in the lab], but you can’t get enough of them to do human trials,” Tremblay notes. “Researchers using primary [original] satellite cells are not considering the practical aspects in humans.”

Although Tremblay has considered using cells other than myoblasts for transplantation, as other investigators are doing, for now he’s persevering with myoblasts. “There’s no perfect treatment for Duchenne dystrophy. I think we have to avoid all jumping on the same bandwagon.”

Looking in blood vessels

Johnny Huard says it makes sense that stem cells would be located in blood vessels.

“If you tell me you want to see stem cells somewhere, I would say look in the blood vessels,” says Johnny Huard, a professor in the Department of Orthopaedic Surgery and Molecular Genetics and Biochemistry at the University of Pittsburgh. Huard received several MDA grants between 1996 and 2005.

“During development, they’re the first cells to come in. It’s probably one of the best places to put stem cells. If you look at a muscle biopsy, you see muscle fibers, but they’re surrounded by thousands of blood vessels. The vessels are so close to the fibers that you almost need an electron microscope [to distinguish them]. That suggests to me that there may be the origin of a lot of stem cells.”

Huard says there are only a few tissues, such as cartilage, that don’t have blood vessels, and those are tissues that can’t repair themselves.

Recently, with Bruno Peault, a professor in the Departments of Pediatrics and Cell Biology at the University of Pittsburgh, and others, Huard identified cells in the blood-vessel walls in human muscle tissue that have both myogenic (muscle-making) characteristics and also resemble the endothelial cells that form the lining of blood vessels. He’s calling these “myoendothelial” cells.

“We believe these cells are in-between blood vessels and muscles and that they’re not committed to one or the other,” Huard says.

Huard believes his myoendothelial cells are related to or even may be the same as a very exciting type of cell called “mesoangioblasts” that were identified by an Italian research group headed by Giulio Cossu at the Istituto Scientifico San Raffaele in Milan.

Cossu, who received MDA support from 2004 to 2007, found mesoangioblasts in mouse, rat and dog blood-vessel walls.

When he injected them into an artery in mice with limb-girdle MD because of an alpha-sarcoglycan deficiency (the human disease is called LGMD 2D), he saw widespread distribution and survival of the donor cells.

In 2006, Cossu’s group showed that mesoangioblasts injected into the arteries of dogs with DMD led to extensive dystrophin production and even some functional recovery of the muscles.

Last year, Cossu showed that similar cells could be isolated from blood vessels in human muscles. Cossu, a conservative scientist, doesn’t yet want to call these mesoangioblasts, because he’s not sure they’re the same as the animal cells, so his group calls them pericytes.

But there’s evidence that mesoangioblasts and pericytes are in fact the same cells. When transplanted into dystrophin-deficient mice (with their immune systems genetically altered to accept human cells), the pericytes generated numerous dystrophin-producing muscle fibers. The mice similarly benefited from pericytes taken from DMD patients and then modified to carry functional dystrophin genes.

The exact relationship of mesoangioblasts and pericytes to the myoendothelial cells identified by Huard, Peault and others remains unknown.

“I will say for sure they are related to mesoangioblasts,” Huard says of his myoendothelial cells. He says myoendothelial cells may be the “parents” of other muscle progenitor cells, but he doesn’t know yet. “We don’t know which is the first,” he says.

Huard also wants to find out whether myoendothelial cells are present in DMD-affected muscles. “I’m pretty sure they will be,” he says, “but I want to make sure they’re there.” The constant repair process that goes on in DMD could exhaust the supply of these cells, he says. “This population might be worn out. That’s something we’re going to be looking at.”

Huard hopes to begin collaborating with Italian researchers to see whether these are in fact the same as his myoendothelial cells or are their close relatives. In either case, he says, “it makes sense that the blood vessels may be very important. This is my view, but a lot of people in the field seem to share that view at the moment.”

A flexible and dynamic place

“Having been one of the people who started early with myoblast transfer 17 years ago, I can say it was a very naive approach,” says Emanuela Gussoni, assistant professor of pediatrics at Harvard Medical School and Children’s Hospital in Boston. “The cells were just stuck in there.”

Researchers in the laboratory of Emanuela Gussoni have found that the protein BMP4 is abundant when cells proliferate and is blocked by gremlin when cells mature.

(MDA has supported four of Gussoni’s projects since 1992. She’s currently receiving support for her work on identifying molecular signals that affect muscle.)

Gussoni says it’s now clear that muscle tissue is a “flexible and dynamic” place, where there’s “a mixture of things that happen. There are a lot of different signals being expressed at the same time or at different times. There’s probably a gradient of different things that push cells to go one way or another way,” depending on what’s needed at a particular location or time, she says.

Some years ago, Gussoni’s group separated muscle-making cells into two groups, which they called the main and side populations, or MP and SP cells. It’s the SP cells that are Gussoni’s main focus.

“SP cell numbers seem to decrease with age,” she says. “There are fewer SP cells the older you get.” Her hypothesis is that SP cells, located in the spaces between muscle fibers (not right next to the fibers, where satellite cells are), can sometimes give rise to satellite cells. They also secrete proteins that change the immediate environment according to what’s needed.

Her group has identified some of these proteins and is particularly interested in two of them, known as BMP4 and gremlin. BMP4 is probably released when there’s a need for cells to proliferate (multiply). Gremlin, which sticks to BMP4 and blocks it, probably is released when cells need to differentiate (mature), Gussoni says.

These processes are often found in varying balances in different tissues and are mutually exclusive. Cells either divide and multiply, or they differentiate into specialized cells, such as muscle or bone. Says Gussoni, “It’s viewing the muscle as a dynamic structure, something that is not just one thing happening at one point. Probably we’ll discover more and more of these proteins.”

At any given time, she thinks, some muscle-making cells (possibly satellite cells) in the vicinity are responding to BMP4 and dividing, while others are starting to differentiate because gremlin is blocking BMP4.

Gussoni says SP cells are “probably precursors of satellite cells to a certain extent. Whether they can give rise to other muscle progenitor cells is not known. We’re trying to address that question.”

The descendants of SP cells can turn into different precursors, Gussoni says, and one of their options is to become muscle precursors. “We need to see if all these precursors arise from one or from multiple places,” she says. SP cells, she believes from studies performed in the laboratory of her longtime Harvard colleague Louis Kunkel, could actually be made up of several subgroups, each of which has the potential to form a different tissue. If that’s the case, she says, “we want to separate out the ones that make muscle.”

Cell transplantation is still worth pursuing, Gussoni believes. Inserted genes may or may not stick around for long, she notes (this is especially true if they’re not integrated into a chromosome), but “cells give material to correct the muscle for a long time.”

Reprogramming cells ‘all the way back’

Margaret (Peggy) Goodell, an MDA grantee at Baylor College of Medicine in Houston, where she’s a professor and director of the Stem Cells and Regenerative Medicine Center, found several years ago that cells from bone marrow could at times contribute to muscle repair and regeneration.

Peggy Goodell is continuing to work on bone marrow cells but is also turning her attention to induced pluripotent stem cells.

“I found they did not contribute to satellite cells, but they occasionally fused with muscle fibers, under certain conditions, such as dystrophin deficiency and injury,” she notes.

“The basic principle is there,” she says, “but we need to enhance the efficiency.” The contribution of bone marrow to muscle is miniscule but potentially could be increased, she thinks.

“I haven’t given up on bone marrow cells completely,” Goodell says. “I’m still working on them in the lab. The fact that muscle cells are fused giant cells makes the whole idea kind of work.” (Muscle fibers are made of fused myoblasts whose cell membranes have disappeared.)

Lately, however, Goodell has turned her attention elsewhere, toward findings from three separate labs announced this past summer showing that skin cells from mice can be turned back into an embryonic state in the laboratory. These cells are now known as “induced pluripotent [having multiple potentials] stem cells,” or IPS cells.

If the same can be accomplished using human skin cells, the implications are far-reaching, Goodell says. “I think this has the potential to revolutionize medicine in general and personalize medicine and cell therapy for a variety of diseases,” she says. (Findings released at press time suggest that human skin cells can also be reprogrammed to become IPS cells.)

The new procedure, if successful in humans, could overcome ethical objections to harvesting stem cells from human embryos, because no embryos or even eggs are required.

“All the limitations we have for getting cells [from donors] would disappear,” Goodell says. Skin cells are much easier to donate than muscle cells or bone marrow. And, she says, “these cells grow infinitely.” If you wanted to use a patient’s own cells, she says, “you could genetically modify them, perhaps even replace a defective gene instead of just adding a gene. You could check the cell and make sure it’s OK before reinjecting it.”

She says it will be important to find out not only if the techniques that worked in mice will work in humans, but whether normal muscle cells can be grown from the IPS cells. It’s also crucial to figure out how far along the path toward becoming muscle an IPS cell should be before it’s transplanted into a patient.

“It seems sensible to partially differentiate cells before putting them into someone,” Goodell says, noting that undifferentiated embryonic cells can form tumors. “But I think putting them in as [completely] muscle-differentiated cells is too difficult. You need some kind of muscle progenitor.”

The procedure would be labor-intensive, Goodell admits, since it would require isolating and culturing cells from each individual patient, but “not prohibitively labor-intensive.” It would be expensive, she notes, to tailor the procedure for each person, but that’s done for bone marrow transplants now.

Goodell is thinking about switching her focus from bone marrow cells to IPS cells. “Maybe it’s easier to reprogram cells all the way back first,” she says. “Then you’re driving them all the way in one direction rather than ‘sideways,’ as you are in going from a blood cell to a muscle cell.”

Local versus systemic delivery

If you’re trying to repair a few inches of cardiac tissue damage after a heart attack or replace a few brain cells lost because of a stroke, it’s clear that you want to deliver the replacement cells locally, or right at the site of the problem. They’d be wasted or possibly even cause trouble if they ended up anywhere else.

But in muscular dystrophy and other neuromuscular diseases, the damage is widespread, leading scientists and physicians to think about ways to deliver the cells to the whole system (systemically) through the circulation, either intravenously (by vein) or intraarterially (by artery).

When Cossu’s group injected mesoangioblasts intra-arterially into dystrophin-deficient dogs and alpha-sarcoglycan-deficient mice, they apparently exited the vascular system into dystrophic muscle fibers in great numbers.

And in a recent study, Lou Kunkel at Children’s Hospital in Boston, Jeffrey Chamberlain at the University of Washington-Seattle, and others found that muscle SP cells injected into an artery in dystrophin-deficient mice fared much better than the same cells injected intravenously.

Jacques Tremblay

But direct intramuscular injection also has its advocates, perhaps most notably Jacques Tremblay. Systemic delivery “has advantages, but it also has problems,” Tremblay says. Cells injected into the bloodstream will get into the liver, kidneys and lungs, he says, where their potential to do harm isn’t yet known.

As for the pain associated with numerous intramuscular injections, Tremblay says local anesthesia takes care of most of it. “It’s really not too bad,” he says, noting that one patient told him the experience was no worse than going to the dentist. “They normally feel the pain only after the limit of the local anesthesia. Then they take Tylenol,” he says.

But Gussoni isn’t sure. “I’m no physician, but I am a mom,” she says. “If I had a kid who had to have millions of injections in their muscles, I don’t know. It’s not the most practical way to cure these kids,” she says, noting that injecting into the heart or diaphragm pose significant challenges. “I can’t see that as being the rescue for muscular dystrophy,” she says, “although I can see it helping, together with other things.”

Systemic delivery of muscle progenitor cells has not yet been tested in humans.

Still work to do

Everyone agrees that some of the hurdles blocking successful muscle-cell transplantation have been cleared since the early 1990s, and there’s a much greater understanding now of how muscle repair occurs and what kinds of cells contribute to it. But not everyone agrees on what conclusions to draw from this progress.

Tremblay says he’s impressed with some of the results researchers are getting with recently identified muscle progenitor cells, such as mesoangioblasts. “Cossu has to be supported for that,” he says, but he’ll continue pursuing myoblasts and, for now anyway, intramuscular delivery.

Gussoni says she isn’t ready to propose human trials for SP cells yet. “In the past few years, a lot of exciting things have come out,” she says. “We have made progress, but we don’t know as much as hematologists know about their cells, and we don’t know how to use SP cells clinically. It’s crucial to be able to expand muscle SP cells in order for them to be clinically useful. And it will be important to define the different muscle-cell precursors at various stages and at different times. We still have work to do in these directions.”

She says she’s “totally open to any kind of cell that will help repair muscle efficiently. If we can do that with muscle SP cells, that’s great. If it can be done with other cells, that’s great too.”

Should families bank cord blood?

Somewhere between the completely undifferentiated (immature and unspecialized) cells of an embryo and the cells of a mature organism lies another type of cell: those present in the umbilical cord at the time a baby is born.

“Cord blood stem cells are pluripotent, which means they can become many types of cells, and they respond to environmental cues in the body,” says David Harris, a professor of microbiology and immunology at the University of Arizona in Tucson, and scientific director of the Cord Blood Registry in that city.

“They seem primitive enough to become multiple cell types, yet mature enough to work within the body’s existing framework,” Harris says, noting that embryonic stem cells don’t always take cues from their cellular environment and can form tumors.

The cells found in the umbilical cord are fetal stem cells, Harris says, and they account for about 1 percent of the cells a human baby has when it’s born. Within 24 hours, he says, that number drops to 0.1 percent, as the fetal cells quickly mature.

Capturing and saving the fetal cells before they mature into specialized cell types could provide a “foundation for regenerative medicine” for the baby and close relatives, Harris says. “For most applications in regenerative medicine, you want your own cells,” he notes. (Of course, if there’s a genetic defect in your own cells, they’ll have to be genetically modified before they can provide benefit.)

They’re being tested in clinical trials to restore blood flow in arterial disease, and they’re being contemplated for use to restore the cornea in the eye, re-establish insulin-producing cells in the pancreas, and repair bone and cartilage.

The possibility that cord blood cells could be used to treat muscular dystrophy is real, Harris believes, and he foresees a time when a sibling’s normal cord blood cells might be given to an affected fetus early in its development to prevent the disease.

But is banking cord blood at this time a worthwhile investment?

Tucson’s Cord Blood Registry (www.cordblood.com) charges about $2,000 for the initial collection, transport, cell processing and storage of a newborn’s cord blood cells, as well as $125 a year for continued storage.

Viacord of Cambridge, Mass. (www.viacord.com), which provides very similar services, has almost identical pricing.

Viacord warns potential clients that “the odds that a family member without a defined risk will need to use their child’s umbilical cord blood are low” and that there’s no guarantee of therapeutic success.

But families who know they have a genetic condition that could one day be treated by umbilical cord blood cells have more reason to consider the procedure.

“I tell people that there isn’t a therapy for a neuromuscular disease based on cord blood stem cells available now,” says Sharon Hesterlee, MDA’s vice president of translational research, “and that it’s difficult to predict what types of stem cells might be used for this purpose.”

Having to genetically modify the cord blood cells adds “a layer of complication to the picture” that isn’t there for leukemia or other nongenetic disorders, she cautions.

“People just need to understand that it’s not a sure thing,” Hesterlee says. “However, I think if a family can afford to and is willing to bank cord blood cells knowing that it’s a long shot, they should go ahead.”